Our goal is to elucidate the regulatory mechanisms of integrin function. As transmembrane receptors, integrins physically connect a cell to another cell or to the extracellular matrix and biochemically transduce signals bidirectionally across the membrane. Understanding the inner workings and regulations of integrins is important because their dysfunction and dysregulation contribute to a wide variety of diseases. The adhesive and signaling activities of integrins depend on their conformations. Applied force can regulate adhesion, signaling, and conformational changes of integrins. In the past grant cycle, we investigated the mechanically-regulated interaction kinetics of single-bonds of integrins 1L22, 1421 and 1521 with their respective ligands, intercellular adhesion molecule 1, vascular adhesion molecule 1 and fibronectin. In this renewal application, we will first directly visualize and characterize the bending and unbending conformational change dynamics of three integrins, 1L22, 1421, and 1521, at the single-molecule level, under controlled forces on living cells in real-time. Using (un)bending measurements as readouts, we will next examine the conformational coupling of unbending with leg separation and hybrid domain swing-out. Thus, our proposal will fill the gaps of crystallography and electron microscopy as these approaches can only observe static conformations in the absence of force. In addition to innovation in the research topics, our approaches are also innovative, as we will combine single-molecule experiments and molecular dynamics simulations with mutagenesis to dissect the structural determinants of integrin conformational change dynamics. Finally, we will bridge biophysics with cell biology by relating the structural- and force-dependent conformational changes to the integrin-ligand binding kinetics. The three specific aims are to: 1) characterize the integrin bending and unbending conformational changes and their regulations; 2) examine coupling among three conformational changes and dissect their structural determinants; 3) relate integrin conformational changes and ligand binding to intracellular signaling. The results of this study will advance our understanding of the inner workings of integrins as molecular nano machines and will serve as a foundation for a better understanding of physiological and pathological processes mediated by integrins.